P. gingivalis antigenic composition

ABSTRACT

The present invention provides an antigenic composition, the composition comprising at least one recombinant protein. The recombinant protein comprises at least one epitope. The epitope is reactive with an antibody which is reactive with a polypeptide having the sequence set out in SEQ. ID. NO. 3 or SEQ. ID. NO. 5. The invention also provides methods and compositions for the production of the recombinant protein. Also provided are methods for the diagnosis, treatment and prevention of  P. gingivalis  infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/174,695, filedJun. 18, 2002, which claims the priority benefit of PCT applicationPCT/AU001/01588, filed Dec. 21, 2000 and Australian Application PQ 4859,filed Dec. 24, 1999, the contents of both of which are'incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention provides an oral composition and an antigenic compositionfor use in the suppression of the pathogenic effects of the intra-oralbacterium Porphyromonas gingivalis associated with periodontal diseasebased on recombinant protein and antibodies. It also provides diagnostictests for the presence of P. gingivalis in subgingival plaque samplesand specific anti-P. gingivalis antibodies in sera. Related thereto anddisclosed is a method for preparing r-RgpA44 and r-Kgp39 and derivativesthereof using recombinant DNA techniques. Also disclosed are host cellstransformed with recombinant vectors capable of expressing therecombinant proteins. The recombinant proteins are useful as immunogensin a vaccine formulation for active immunization and can be used togenerate protein-specific antisera useful for passive immunization andas reagents for diagnostic assays.

BACKGROUND OF THE INVENTION

This invention relates generally to recombinant proteins ofPorphyromonas gingivalis, r-RgpA44 and r-Kgp39. The invention alsorelates to pharmaceutical compositions and associated agents based onthese recombinant proteins and derivatives for the detection, preventionand treatment of periodontal disease associated with P. gingivalis.

Periodontal diseases are bacterial-associated inflammatory diseases ofthe supporting tissues of the teeth and range from the relatively mildform of gingivitis, the non-specific, reversible inflammation ofgingival tissue to the more aggressive forms of periodontitis which arecharacterised by the destruction of the tooth's supporting structures.Periodontitis is associated with a subgingival infection of a consortiumof specific Gram-negative bacteria that leads to the destruction of theperiodontium and is a major public health problem. One bacterium thathas attracted considerable interest is P. gingivalis as the recovery ofthis microorganism from adult periodontitis lesions can be up to 50% ofthe subgingival anaerobically cultivable flora, whereas P. gingivalis israrely recovered, and then in low numbers, from healthy sites. Aproportional increase in the level of P. gingivalis in subgingivalplaque has been associated with an increased severity of periodontitisand eradication of the microorganism from the cultivable subgingivalmicrobial population is accompanied by resolution of the disease. Theprogression of periodontitis lesions in non-human primates has beendemonstrated with the subgingival implantation of P. gingivalis. Thesefindings in both animals and humans suggest a major role for P.gingivalis in the development of adult periodontitis.

More recently there has been increasing linkage of priodontal diseaseand cardiovascular disease and therefore a link between P. gingivalisinfection and cardiovascular disease. More information regarding thislinkage can be found in Beck, J D et al. Ann Periodontol. 3: 127-141,1998 and Beck, J. et al. J. Periodontol. 67:1123-37, 1996.

P. gingivalis expresses a range of proteins on its cell surface that arepotential candidates for the development of a vaccine or diagnostic. Amajor group of cell surface proteins expressed by P. gingivalis is agroup of proteinases and associated adhesins. One proteinase designatedArg-gingipain has been disclosed previously by Travis et al. (PCTPublication No. WO 95/07286). These investigators also reported a highmolecular mass form of Arg-gingipain that is encoded by the gene rgpalso disclosed in WO 95/07286. The high molecular mass form ofArg-gingipain consists of the proteinase and several other proteinsproposed to be adhesins. Cell-surface complexes of P. gingivalisconsisting of Arg- and Lys-specific proteinases and adhesins have alsobeen disclosed by Reynolds et al. (PCT/AU96/00673). Neither of thesedisclosures provide teaching regarding the utility of a particularadhesin as a recombinant in the protection of P. gingivalis infection.

SUMMARY OF THE INVENTION

In a first aspect the present invention consists in an antigeniccomposition, the composition comprising at least one recombinantprotein, the recombinant protein comprising at least one epitope, theepitope being reactive with an antibody wherein the antibody is reactivewith a polypeptide having the sequence set out in SEQ. ID. NO. 3 or SEQ.ID. NO. 5.

In a further preferred embodiment the antigenic composition comprises arecombinant protein having a sequence selected from the group consistingof SEQ. ID. NO. 3, residues 1-184 of SEQ. ID. NO. 3, residues 1-290 ofSEQ. ID. NO. 3, residues 65-184 of SEQ. ID. NO. 3, residues 65-290 ofSEQ. ID. NO. 3, residues 65-419 of SEQ. ID. NO. 3, residues 192-290 ofSEQ. ID. NO. 3, residues 192-419 of SEQ. ID. NO. 3, residues 147-419 ofSEQ. ID. NO. 3, SEQ. ID. NO. 5 and SEQ. ID. NO. 6.

As will be noted from a comparison of SEQ. ID. NO. 3 and SEQ. ID. NO. 5these polypeptides are identical over a substantial portion of theirsequence.

In another preferred embodiment the antigenic composition furthercomprises an adjuvant.

In yet another preferred embodiment the recombinant protein is achimeric or a fusion protein. Where the recombinant protein is achimeric or a fusion protein it is preferred that protein include asequence selected from the group consisting of SEQ. ID. NO. 3, residues1-184 of SEQ. ID. NO. 3, residues 1-290 of SEQ. ID. NO. 3, residues65-184 of SEQ. ID. NO. 3, residues 65-290 of SEQ. ID. NO. 3, residues65-419 of SEQ. ID. NO. 3, residues 192-290 of SEQ. ID. NO. 3, residues192-419 of SEQ. ID. NO. 3, residues 147-419 of SEQ. ID. NO. 3, SEQ. ID.NO. 5 and SEQ. ID. NO. 6. An example of such a chimeric or a fusionprotein is set out in SEQ. ID. NO. 4.

In a second aspect the present invention consists in a composition, thecomposition comprising at least one antibody, the antibody being raisedagainst the antigenic composition of the first aspect of the presentinvention.

In a third aspect the present invention consists in a recombinantprokaryotic or eucaryotic cell, the recombinant cell comprising a DNAsequence selected from the group consisting of SEQ. ID. NO. 1,nucleotides 1-1257 of SEQ. ID. NO. 1, nucleotides 1-552 of SEQ. ID. NO.1, nucleotides 1-870 of SEQ. ID. NO. 1, nucleotides 193-552 of SEQ. ID.NO. 1, nucleotides 193-870 of SEQ. ID. NO. 1, nucleotides 193-1257 ofSEQ. ID. NO. 1, nucleotides 574-870 of SEQ. ID. NO. 1, nucleotides574-1257 of SEQ. ID. NO. 1, nucleotides 439-1257 of SEQ. ID. NO. 1, SEQ.ID NO. 7, SEQ. ID. NO. 8 and sequences which hybridises thereto understringent conditions operatively linked to at least one regulatoryelement.

As used herein, stringent conditions are those that (1) employ low ionicstrength and high temperature for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate/0.1% NaDodSO₄ at 50° C.; (2) employ duringhybridisation a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide,5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmonsperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in0.2×SSC and 0.1% SDS.

In a further aspect the present invention consists in a method ofpreventing or reducing the incidence or severity of P. gingivalisinfection in a subject, the method comprising administering to thesubject the antigenic composition of the first aspect of the presentinvention.

Given the increasing linkage of periodontal disease with cardiovasculardisease (CVD) and the possible link therefore of P. gingivalis infectionand CVD the antigenic composition of the first aspect of the presentinvention may also be used in a prophylactic therapy to reduce theincidence or severity of CVD or as an adjunct in treating CVD.

An important form of the invention is a vaccine based on the r-RgpA44and/or r-Kgp39 proteins or peptides and suitable adjuvant delivered bynasal spray, orally or by injection to produce a specific immuneresponse against the RgpA44 and/or r-Kgp39 protein. A vaccine can alsobe based upon a recombinant component of the RgpA44 and/or Kgp39 genesegment incorporated into an appropriate vector and expressed in asuitable transformed host (e.g. E. coli, Bacillus subtilis,Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells)containing the vector. Component protein, peptides, and oligopeptideswith immunogenic epitopes from the RgpA44 and/or Kgp39 protein, can beused as immunogens in various vaccine formulations in the prevention ofperiodontal diseases. Additionally, according to the present invention,the RgpA44 and/or Kgp39 proteins and related peptides or chimerasproduced may be used to generate P. gingivalis antisera useful forpassive immunization against periodontal disease and infections causedby P. gingivalis.

According to one embodiment of the present invention, using recombinantDNA techniques the gene segment encoding RgpA44 and/or Kgp39, or genefragments encoding one or more peptides or chimeras having immunogenicepitopes, is incorporated into an expression vector, and the recombinantvector is introduced into an appropriate host cell thereby directing theexpression of these sequences in that particular host cell. Theexpression system, comprising the recombinant vector introduced into thehost cell, can be used (a) to produce r-RgpA44 and/or r-Kgp39 proteins,related peptides, oligopeptides or chimeras which can be purified foruse as an immunogen in vaccine formulations; (b) to produce RgpA44and/or Kgp39 protein, related peptides, oligopeptides and chimeras to beused as an antigen for diagnostic immunoassays or for generating P.gingivalis-specific antisera of therapeutic and/or diagnostic value; (c)or if the recombinant expression vector is a live virus such as vacciniavirus, the vector itself may be used as a live or inactivated vaccinepreparation to be introduced into the host's cells for expression ofRgpA44 and/or Kgp39 or immunogenic peptides or oligopeptides or chimericpeptides; (d) for introduction into live attenuated bacterial cells orgenetically engineered commensal intra-oral bacteria which are used toexpress RgpA44 and/or Kgp39 protein, related peptides or oligopeptidesor chimeras to vaccinate individuals; (e) or for introduction directlyinto an individual to immunize against the encoded and expressed RgpA44protein, related peptides, or oligopeptides or chimeras. In particularthe recombinant bacterial vaccine can be based on a commensal inhabitantof the human oral cavity or animal if the vaccine is to preventperiodontal disease in animals. The recombinant bacterial vaccineexpressing P. gingivalis RgpA44 and/or Kgp39 can be used to colonise theoral cavity, supragingival or subgingival plaque. The intra-oralbacterium can be isolated from the patient with periodontitis andgenetically engineered to express the r-RgpA44 and/or r-Kgp39,components, peptides or chimeras. The r-RgpA44 and/or r-Kgp39 proteinwill stimulate the mucosal-associated lymphoid tissues (NIALT) toproduce specific antibody to P. gingivalis.

RgpA44 and/or Kgp39 proteins, peptides, oligopeptides, chimeric peptidesand constructs containing epitopes can be used as immunogens inprophylactic and/or therapeutic vaccine formulations against pathogenicstrains of P. gingivalis, whether the immunogen is chemicallysynthesized, purified from P. gingivalis, or purified from a recombinantexpression vector system. Alternatively, the gene segment encodingRgpA44 and/or Kgp39, or one or more gene fragments encoding peptides oroligopeptides or chimeric peptides, may be incorporated into a bacterialor viral vaccine comprising recombinant bacteria or virus which isengineered to produce one or more specific immunogenic epitopes ofRgpA44 and/or Kgp39, or in combination with immunogenic epitopes ofother pathogenic microorganisms. In addition, the gene encoding RgpA44and/or Kgp39 or one or more gene fragments encoding RgpA44 and/or Kgp39peptides or oligopeptides or chimeric peptides, operatively linked toone or more regulatory elements, can be introduced directly into humansto express protein, peptide, oligopeptides or chimeric peptides relatingto the RgpA44 and/or Kgp39 to elicit a protective immune response. Avaccine can also be based upon a recombinant component of normal ormutated RgpA44 and/or Kgp39 incorporated into an appropriate vector andexpressed in a suitable transformed host (e.g. E. coli, Bacillussubtilis, Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells)containing the vector. The vaccine can be based on an intra-oralrecombinant bacterial vaccine, where the recombinant bacteriumexpressing the P. gingivalis RgpA44 and/or Kgp39 is a commensalinhabitant of the oral cavity.

In another aspect, the invention provides nucleotide sequences codingfor the recombinant protein and functional equivalents of saidnucleotide sequences and nucleic acid probes for said nucleotidesequences.

The invention also includes within its scope various applications anduses of the above nucleotides and recombinant products includingchimeric recombinant polypeptides. In particular, the invention providesantibodies raised against the r-RgpA44 or r-Kgp39, herein calledanti-r-RgpA44 antibodies and anti-r-Kgp39 antibodies, respectively; andantibodies to the polypeptides, oligopeptides and chimeric peptides. Theantibodies may be polyclonal or monoclonal. The antibodies may beblended into oral compositions such as toothpaste, mouthwash,toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums,dental pastes, gingival massage creams, gargle tablets, dairy productsand other foodstuffs. The recombinant polypeptides, oligopeptides andchimeric peptides may also be used as immunogens in prophylactic and/ortherapeutic vaccine formulations.

In another aspect the invention provides a method of diagnosis for thepresence of P. gingivalis characterised by the use of any one or acombination of an antibody, antigen or nucleic acid probe ashereinbefore defined comprising the application of known techniquesincluding for example, enzyme linked immunosorbent assay.

The invention also provides diagnostic kits comprising antibodies,antigens and/or nucleic acid probes as hereinbefore defined.

The invention also provides a method of treatment of a patient eithersuffering from P. gingivalis infection comprising active vaccination ofsaid patient with a vaccine as hereinbefore defined and/or passivevaccination of said patient with an antibody as hereinbefore defined.

DETAILED DESCRIPTION OF THE INVENTION Figure Legends

FIG. 1 shows the results obtained in Example 1.

FIG. 2 shows the results of the full length recombinant 44 kD protein, 2fragments of the 44 kD protein (Fragment 4; residues 65-290 and fragment6; residues 192-290), a control recombinant protein R2 and Formalinkilled whole P. gingivalis (FK-33277) in the mouse abscess model.

FIG. 3. Flow cytometric analysis of P. gingivalis cells reacted with (A)PBS/FA, (B) normal mouse serum, (C) P. gingivalis whole cell antisera,(D) recombinant Pg44 antisera, (E) Fragment 4 antisera (r-44 kDaresidues 65-290) (F) Fragment 6 antisera (r-44 kDa residues 192-290) (G)Chimeric r-44-Pg33 protein antisera.

FIG. 4. Binding of the RgpA-Kgp specific anti-sera to recombinantproteins. The recombinant proteins were coated at 5 μg/ml and probedwith anti-RgpA-Kgp specific anti-sera: recombinant Kgp39 protein (-♦-),recombinant Kgp39 fragment (-▴-), RgpA-Kgp complex (--), and control(-X-). Bound antibody was detected using a 1:4000 dilution of Goatanti-Rabbit HRP, and ELISA plates were read using a Labsystems iEMSmicroplate reader at 415 nm.

FIG. 5. Binding of recombinant Kgp39 protein to a variety of matrixproteins. The matrix proteins were coated at 5 μg/ml and probed withrecombinant protein which was then probed with anti-RgpA-Kgp complexspecific anti-sera: Collagen type V (-♦-), Fibrinogen (-▪-), Hemoglobin(-▴-), and control (-X-). Bound antibody was detected using a 1:4000dilution of Goat anti-Rabbit HRP conjugate, and ELISA plates were readusing a Labsystems iEMS reader at 415 nm.

FIG. 6. Binding of recombinant Kgp39 protein fragment to a variety ofmatrix proteins. The matrix proteins were coated at 5 μg/ml and probedwith recombinant protein which was then probed with anti-RgpA-Kgpcomplex specific anti-sera: Collagen type V (-♦-), Fibrinogen (-▪-),Hemoglobin (-▴-), and control (-X-). Bound antibody was detected using a1:4000 dilution of Goat anti-Rabbit HRP conjugate, and ELISA plates wereread using a Labsystems iEMS reader at 415 nm.

In order that the nature of the present invention may be more clearlyunderstood preferred forms thereof will be described with reference tothe following Examples.

The intra-oral bacterium Porphyromonas gingivalis contains on itssurface a proteinase-adhesin complex encoded by the genes rgpA and kgp.The recombinant 44 kDa adhesin (r-RgpA44) of this proteinase-adhesincomplex protects against P. gingivalis challenge in a mouse abscessmodel whereas other recombinant proteins from the rgpA gene do not. Thegene segment encoding the 44 kDa adhesin domain RgpA44 or Kgp39 can becloned into an appropriate expression system to produce the recombinantprotein, r-RgpA44 or r-Kgp39. The purified r-RgpA44 or r-Kgp39 proteincan then be used to generate antibodies using standard techniques. Theanimals used for antibody generation can be rabbits, goats, chickens,sheep, horses, cows etc. When a high antibody titre against the r-RgpA44or r-Kgp39 protein is detected by immunoassay the animals are bled oreggs or milk are collected and the serum prepared and/or antibodypurified using standard techniques or monoclonal antibodies produced byfusing spleen cells with myeloma cells using standard techniques. Theantibody (immunoglobulin fraction) may be separated from the culture orascites fluid, serum, milk or egg by salting out, gel filtration, ionexchange and/or affinity chromatography, and the like, with salting outbeing preferred. In the salting out method the antiserum or the milk issaturated with ammonium sulphate to produce a precipitate, followed bydialyzing the precipitate against physiological saline to obtain thepurified immunoglobulin fraction with the specific anti-r-RgpA44 oranti-r-Kgp39. The preferred antibody is obtained from the equineantiserum and the bovine antiserum and milk. In this invention theantibody contained in the antiserum and milk obtained by immunising theanimal with the r-RgpA44 or r-Kgp39 protein or peptide is blended intothe oral composition. In this case the antiserum and milk as well as theantibody separated and purified from the antiserum and milk may be used.Each of these materials may be used alone or in combination of two ormore. Antibodies against the r-RgpA44 or r-Kgp39 can be used in oralcompositions such as toothpaste and mouthwash. The anti-r-RgpA44 oranti-rKgp39 antibodies can also be used for the early detection of P.gingivalis in subgingival plaque samples by a chairside Enzyme LinkedImmunosorbent Assay (ELISA).

For oral compositions it is preferred that the amount of the aboveantibodies administered is 0.0001-50 g/kg/day and that the content ofthe above antibodies is 0.0002-10% by weight preferably 0.002-5% byweight of the composition. The oral composition of this invention whichcontains the above-mentioned serum or milk antibody maybe prepared andused in various forms applicable to the mouth such as dentifriceincluding toothpastes, toothpowders and liquid dentifrices, mouthwashes,troches, periodontal pocket irrigating devices, chewing gums, dentalpastes, gingival massage creams, gargle tablets, dairy products andother foodstuffs. The oral composition according to this invention mayfurther include additional well-known ingredients depending on the typeand form of a particular oral composition.

In certain highly preferred forms of the invention the oral compositionmay be substantially liquid in character, such as a mouthwash or rinse.In such a preparation the vehicle is typically a water-alcohol mixturedesirably including a humectant as described below. Generally, theweight ratio of water to alcohol is in the range of from about 1:1 toabout 20:1. The total amount of water-alcohol mixture in this type ofpreparation is typically in the range of from about 70 to about 99.9% byweight of the preparation. The alcohol is typically ethanol orisopropanol. Ethanol is preferred.

The pH of such liquid and other preparations of the invention isgenerally in the range of from about 4.5 to about 9 and typically fromabout 5.5 to 8. The pH is preferably in the range of from about 6 toabout 8.0, preferably The pH can be controlled with acid (e.g. citricacid or benzoic acid) or base (e.g. sodium hydroxide) or buffered (aswith sodium citrate, benzoate, carbonate, or bicarbonate, disodiumhydrogen phosphate, sodium dihydrogen phosphate, etc).

Other desirable forms of this invention, the oral composition may besubstantially solid or pasty in character, such as toothpowder, a dentaltablet or a dentifrice, that is a toothpaste (dental cream) or geldentifrice. The vehicle of such solid or pasty oral preparationsgenerally contains dentally acceptable polishing material. Examples ofpolishing materials are water-insoluble sodium metaphosphate, potassiummetaphosphate, tricalcium phosphate, dihydrated calcium phosphate,anhydrous dicalcium phosphate, calcium pyrophosphate, magnesiumorthophosphate, trimapesium phosphate, calcium carbonate, hydratedalumina, calcined alumina, aluminum silicate, zirconium silicate,silica, bentonite, and mixtures thereof. Other suitable polishingmaterial include the particulate thermosetting resins such as melamine-,phenolic, and urea-formaldehydes, and cross-linked polyepoxides andpolyesters. Preferred polishing materials include crystalline silicahaving particle size of up to about 5 microns, a mean particle size ofup to about 1. 1 n-Acrons, and a surface area of up to about 50,000cm²/gm, silica gel or colloidal silica, and complex amorphous alkalimetal aluminosilicate.

When visually clear gels are employed, a polishing agent of colloidalsilica, such as those sold under the trademark SYLOID as Syloid 72 andSyloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metalalumino-silicate complexes are particularly useful since they haverefractive indices close to the refractive indices of gellingagent-liquid (including water and/or humectant) systems commonly used indentifrices.

Many of the so-called “water insoluble” polishing materials are anionicin character and also include small amounts of soluble material. Thus,insoluble sodium metaphosphate may be formed in any suitable manner asillustrated by Thorpe's Dictionary of Applied Chemistry, Volume 9, 4thEdition, pp. 510-511. The forms of insoluble sodium metaphosphate knownas Madrell's salt and Kurrol's salt are further examples of suitablematerials. These metaphosphate salts exhibit only a minute solubility inwater, and therefore are commonly referred to as insolublemetaphosphates (IMP). There is present therein a minor amount of solublephosphate material as impurities, usually a few percent such as up to 4%by weight. The amount of soluble phosphate material, which is believedto include a soluble sodium trimetaphosphate in the case of insolublemetaphosphate, may be reduced or eliminated by washing with water ifdesired. The insoluble alkali metal metaphosphate is typically employedin powder form of a particle size such that no more than 1% of thematerial is larger than 37 microns.

The polishing material is generally present in the solid or pastycompositions in weight concentrations of about 10% to about 99%.Preferably, it is present in amounts from about 10% to about 75% intoothpaste, and from about 70% to about 99% in toothpowder. Intoothpastes, when the polishing material is silicious in nature, it isgenerally present in amount of about 10-30% by weight. Other polishingmaterials are typically present in amount of about 30-75% by weight.

In a toothpaste, the liquid vehicle may comprise water and humectanttypically in an amount ranging from about 10% to about 80% by weight ofthe preparation. Glycerine, propylene glycol, sorbitol and polypropyleneglycol exemplify suitable humectants/carriers. Also advantageous areliquid mixtures of water, glycerine and sorbitol. In clear gels wherethe refractive index is an important consideration, about 2.5-30% w/w ofwater, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitolare preferably employed.

Toothpaste, creams and gels typically contain a natural or syntheticthickener or gelling agent in proportions of about 0.1 to about 10,preferably about 0.5 to about 5% w/w. A suitable thickener is synthetichectorite, a synthetic colloidal magnesium alkali metal silicate complexclay available for example as Laponite (e.g. CP, SP 2002, D) marketed byLaporte Industries Limited. Laponite D is, approximately by weight58.00% SiO₂, 25.40% MgO, 3.05% Na₂O, 0.98% Li₂O, and some water andtrace metals. Its true specific gravity is 2.53 and it has an apparentbulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gumtragacanth, starch, polyidnylpyrrolidone, hydroxyethylpropylcellulose,hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose,hydroxyethyl cellulose (e.g. available as Natrosol), sodiumcarboxymethyl cellulose, and colloidal silica such as finely groundSyloid (e.g. 244). Solubilizing agents may also be included such ashumectant polyols such propylene glycol, dipropylene glycol and hexyleneglycol, cellosolves such as methyl cellosolve and ethyl cellosolve,vegetable oils and waxes containing at least about 12 carbons in astraight chain such as olive oil, castor oil and petrolatum and esterssuch as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparationsare to be sold or otherwise distributed in suitable labeled packages.Thus, a jar of mouthrinse will have a label describing it, in substance,as a mouthrinse or mouthwash and having directions for its use; and atoothpaste, cream or gel will usually be in a collapsible tube,typically aluminum, lined lead or plastic, or other squeeze, pump orpressurized dispenser for metering out the contents, having a labeldescribing it, in substance, as a toothpaste, gel or dental cream.

Organic surface-active agents are used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, nonionic or ampholytic in nature which does notdenature the antibody of the invention, and it is preferred to employ asthe surface-active agent a detersive material which imparts to thecomposition detersive and foaming properties while not denaturing theantibody. Suitable examples of anionic surfactants are water-solublesalts of higher fatty acid monoglyceride monosulfates, such as thesodium salt of the monosulfated monoglyceride of hydrogenated coconutoil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate,alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higheralkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propanesulfonate, and the substantially saturated higher aliphatic acyl amidesof lower aliphatic amino carboxylic acid compounds, such as those having12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and thelike. Examples of the last mentioned amides are N-lauroyl sarcosine, andthe sodium, potassium, and ethanolamine salts of N-lauroyl, N-myristoyl,or N-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. The use of these sarconite compoundsin the oral compositions of the present invention is particularlyadvantageous since these materials exhibit a prolonged marked effect inthe inhibition of acid formation in the oral cavity due to carbohydratesbreakdown in addition to exerting some reduction in the solubility oftooth enamel in acid solutions. Examples of water-soluble nonionicsurfactants; suitable for use with antibodies are condensation productsof ethylene oxide with various reactive hydrogen-containing compoundsreactive therewith having long hydrophobic chains (e.g. aliphatic chainsof about 12 to 20 carbon atoms), which condensation products(“ethoxamers”) contain hydrophilic polyoxyethylene moieties, such ascondensation products of poly (ethylene oxide) with fatty acids, fattyalcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate)and polypropyleneoxide (e.g. Pluronic materials).

Surface active agent is typically present in amount of about 0.1-5% byweight. It is noteworthy, that the surface active agent may assist inthe dissolving of the antibody of the invention and thereby diminish theamount of solubilizing humectant needed.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavoring or sweetening material may also be employed.Examples of suitable flavoring constituents are flavoring oils, e.g. oilof spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavor and sweetening agents may each or together comprisefrom about 0.1% to 5% more of the preparation.

In the preferred practice of this invention an oral compositionaccording to this invention such as mouthwash or dentifrice containingthe composition of the present invention is preferably applied regularlyto the gums and teeth, such as every day or every second or third day orpreferably from 1 to 3 times daily, at a pH of about 4.5 to about 9,generally about 5.5 to about 8, preferably about 6 to 8, for at least 2weeks up to 8 weeks or more up to a lifetime.

The compositions of this invention can be incorporated in lozenges, orin chewing gum or other products, e.g. by stirring into a warm gum baseor coating the outer surface of a gum base, illustrative of which may bementioned jelutong, rubber latex, vinylite resins, etc., desirably withconventional plasticizers or softeners, sugar or other sweeteners orsuch as glucose, sorbitol and the like.

The composition of this invention also includes targeted deliveryvehicles such as periodontal pocket irrigation devices, collagen,elastin, or synthetic sponges, membranes or fibres placed in theperiodontal pocket or used as a barrier membrane or applied directly tothe tooth root.

The following examples are further illustrative of the nature of thepresent invention, but it is understood that the invention is notlimited thereto. All amounts and proportions referred to herein and inthe appended claims are by weight unless otherwise indicated.

EXAMPLE 1

Cloning and expression of the P. gingivalis proteinase and adhesindomains RgpA45, RgpA44, RgpA27 and RgpA15 in E. coli and testing of therecombinant proteins as a vaccine in the murine abscess model.

TABLE 1 Oligonucleotide primers used for the amplificationof the nucleotide sequences encoding RgpA45, RgpA44, RgpA27 and RgpA15.Recombinant Protein Primers RgpA45Forward 5′-GCGCAGATCTTACACACCGGTAGAGG-3′ (SEQ ID NO.: 9)Reverse 5′-GCGCGTCGACTTAGCGAAGAAGTTCGGGG-3′ (SEQ ID NO.: 10) RgpA44Forward 5′-GCGCCATATGAGCGGTCAGGCCGAGATTGTTCTTG-3′ (SEQ ID NO.: 11)Reverse 5′-GCGCCTCGAGGCGCTTGCCATTGGCCTTGATCTC-3′ (SEQ ID NO.: 12) RgpA27Forward 5′-GCGCGCTAGCGTATACATGGCCAACGAAGCCAAGG-3′ (SEQ ID NO.: 13)Reverse 5′-GCGCAGATCTCTTGATAGCGAGTTTCTC-3′ (SEQ ID NO.: 14) RgpA15Forward 5′-GCGCGCTAGCGTATACATGGCAGACTTCACGGAAACGTTC-3′ (SEQ ID NO.: 15)Reverse 5′-GCGCAGATCTTTTGGCGCCATCGGCTTCCG-3′ (SEQ ID NO.: 16)

Each of the proteinase and adhesin domains of the gene rgpA wereamplified using the primers listed in Table 1, P. gingivalis W50 genomicDNA with Elongase® (Gibco BRI) DNA polymerase and a PC-960 thermalcycler (Corbett Research Technologies). Using the oligonucleotideprimers a PCR was performed essentially as described in the Elongaseinstruction protocol using the following conditions: 25 cycles ofdenaturation (94° C., 30 sec), annealing (50° C., 45 sec), and extension(70° C., 1.5 min). The PCR product was purified using PCR Spinclean®(Progen) and ligated into plasmid vector pGEMT-easy (Promega) andtransformed into competent E. coli JM109 (Promega) following themanufacturers protocols. All procedures were similar for the preparationof the four recombinants so the detailed process for the RgpA44 onlywill be described. Recombinant plasmid pGEMT-easy-RgpA44 DNA wasdigested with NdeI and XhoI to release the insert DNA. Insert DNA wasisolated by agarose gel electrophoresis (0.8%) and purified using theQiafilter gel extraction kit (Qiagen). Purified insert DNA was ligatedinto Qiafilter purified plasmid expression vector pET28a (Novagen) thathad been previously digested with NdeI and XhoI, and the ligationproducts were transformed into the non-expression host, E. coli JM109.The recombinant pET28-RgpA44 plasmid was then transformed into the E.coli expression host, HMS174(DE3) and selected on LB containing 50 μgkanamycin. The r-RgpA44 expressed from pET28a contains a hexahistidinetag fused to the N-terminus of the expressed recombinant protein.r-RgpA44 expression was induced by addition of IPTG and purified bynickel-affinity chromatography. The integrity of the insert ofpET28-RgpA44 was confirmed by DNA sequence analysis.

Expression of Recombinant E. Coli

A single colony transformant was used to inoculate 10 mls ofLuria-Bertani broth containing 50 μg/ml kanamycin at 37° C. until theoptical density (OD₆₀₀) was 1.0. This inoculum was then used toinoculate 500 ml of Terrific broth (containing potassium phosphates and50 μg/ml kanamycin). The OD₆₀₀ of this culture was allowed to reach 2.0before inducing with 0.1 mM IPTG. After a 4.5 hour induction period at37° C. the culture was harvested by centrifuging at 4000 rpm for 20 minat 4° C. and the pellet was stored at −70° C. for the extraction ofinclusion bodies.

Isolation and Solubilisation of Inclusion Bodies

The bacterial pellet was thawed on ice and resuspended in binding buffer(5 mM imidazole, 500 nM NaCl, 20 mM Tris-HCl, pH 7.9), then sonicatedand centrifuged at 20,000×g to collect the inclusion bodies. The pelletwas resuspended in binding buffer and the process of sonication andcentrifugation repeated twice more to release further protein. Thepellet was then resuspended in binding buffer containing 6 M urea andincubated on ice for 2-3 hrs stirring to completely dissolve proteins.Any remaining insoluble material was removed by centrifuging at 39,000×gfor 20 min. The supernatant was filtered through a 0.45 μm membranebefore column purification.

Nickel-Nitrilotriaectic Acid (Ni-NTA) Purification and Refolding ofSolubilised Inclusions

Ni-NTA metal affinity chromatography was used to purify the recombinantproteins via the H₆ tag. Briefly, proteins were batch bound to theequilibrated Ni-NTA resin (Qiagen) which was poured into a small columnand unbound proteins were eluted under gravity. The column was thenwashed with 10 volumes of binding buffer followed by 5 column volumes ofwash buffer (60 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, 6M urea, pH7.9). The bound protein was then eluted in buffer containing 1Mimidazole, 500 mM NaCl, 20 mM Tris-HCl, 6M urea, pH 7.9).

Renaturation Of Recombinant Protein

Fractions eluted off the NI-NTA resin were pooled and refolded by thestep-wise dialysis from 6 M to 3 M to 1.5 M to 0 M Urea contained in thefollowing buffer 0.5 M Tris-HCl, 50 mM NaCl and 8% Glycerol.

Polyacrylamide Gel Electrophoresis and Western Blotting

SDS-PAGE was performed as described by Laemmli. Samples were mixed withan equal volume of 2× sample reducing buffer, boiled for 10 min at 95°C. and ran on Tris-glycine 12% gels (Novex). Molecular weight standards(SeeBlue™) were also purchased from Novex. Western blots were preparedby electroblotting proteins onto nitrocellulose for 1 hr at 100 volts.Membranes were blocked with 1% casein solution before incubating withprimary antibody diluted to 1/1000, washed and incubated with an goatanti-rabbit-BRP conjugate (KPL) washed and developed with TMB membraneperoxidase substrate (KPL).

Antisera

Polyclonal antiserum was raised to the purified recombinant proteins bydosing BALB/c mice with 2×20 μg of recombinant protein in Freundsincomplete adjuvant (Sigma) three weeks apart. Mice were bled one weekafter the second dose and the antiserum generated was used to screenWestern blots of whole cell P. gingivalis W50 run under denaturing,reducing conditions.

The purity of the recombinant proteins was confirmed using MALDI-TOFmass spectrometry and N-terminal sequence analysis.

Murine Lesion Model

Groups of 10 female BALB/c mice (6-8 weeks old) were immunized (20 μg)subcutaneously with each recombinant protein, r-RgpA45, r-RgpA44,r-RgpA27 and r-RgpA15 as well as formalin-killed P. gingivalis cells andE. coli; all emulsified in Incomplete Freunds Adjuvant. Theimmunizations were given at the base of the tail and occurred four weeksand one week prior to challenge with P. gingivalis. Two days prior tochallenge mice were bled from the retrobulbar plexus. BALB/c mice werechallenged with 7.5×10⁹ viable cells of P. gingivalis 33277subcutaneously in the abdomen. Following challenge, mice were examineddaily for the number and size of lesions over a period of seven days.Lesions developed on the abdomen of the mice and the maximum lesion sizein MM² is presented in FIG. 1. Significant reductions in lesion sizewere obtained only with vaccination using formalin-killed whole P.gingivalis cells and the recombinant adhesin r-RgpA44. The otherrecombinant proteins from the rgpA gene did not significantly reducelesion size.

This example demonstrates the superiority of r-RgpA44 over the otherrecombinant proteins from the rgpA gene in protection against P.gingivalis challenge.

EXAMPLE 2

In the previous example it was demonstrated that the recombinant 44 kDaadhesin protected against challenge with P. gingivalis in the mouselesion model. However the full length 44 kDa adhesin when expressed inE. coli was found as inclusion bodies that were only soluble indenaturing solvents. A series of fragments from the 44 kDa adhesin weregenerated in order to improve the solublility of the protein and enhancethe correct folding of the recombinant protein. The oligonucleotideprimers used to construct fragments of the 44 kDa adhesin recombinantprotein are shown in Table 2.

TABLE 2 Oligonucleotide primers used for construction ofthe r-protein vectors Recombinant protein Direction Primers Fragment 1 F5′-GGGAATTCCATGGGTCAGGCCGAGATTGTT-3′ (SEQ ID NO.: 17) Fragment 1 R5′-TCCCTCGAGCTTAACTTCCACGCAATACTC-3′ (SEQ ID NO.: 18) Fragment 2 F5′-GGGAATTCCATGGGTCAGGCCGAGATTGTT-3′ (SEQ ID NO.: 19) Fragment 2 R5′-GGTCAATTGGACTCGAGATATACACAACCATTGCT-3′ (SEQ ID NO.: 20) Fragment 3 F3′-GAGGAATTCAGATCCTTCTTGTTCCCCTAC-3′ (SEQ ID NO.: 21) Fragment 3 R5′-TCCCTCGAGCTTAACTTCCACGCAATACTC-3′ (SEQ ID NO.: 22) Fraament 4 F5′-GAGGAATTCAGATCCTTCTTGTTCCCCTAC-3′ (SEQ ID NO.: 23) Fraament 4 R5′-GGTCAATTGGACTCGAGATATACACAACCATTGCT-3′ (SEQ ID NO.: 24) Fragment 5 F5′-GAGGAATTCAGATCCTTCTTGTTCCCCTAC-3′ (SEQ ID NO.: 25) Fragment 5 R5′-AGGAATTCTCGAGCTTGCCGTTGGCCTTGAT-3′ (SEQ ID NO.: 26) Fragment 6 F5′-GGGAATTCCATGGCGAAGGTATGTAAAGACGTT-3′ (SEQ ID NO.: 27) Fragment 6 R5′-GGTCAATTGGACTCGAGATATACACAACCATTGCT-3′ (SEQ ID NO.: 28) Fragment 7 F5′-GGGAATTCCATGGCGAAGGTATGTAAAGACGTT-3′ (SEQ ID NO.: 29) Fragment 7 R5′-AGGAATTCTCGAGCTTGCCGTTGGCCTTGAT-3′ (SEQ ID NO.: 30)

Using similar methods as described in Example 1, fragments of the 44 kDaadhesin were cloned into pET24b plasmids (Novagen) and expressed in E.coli strain BL21(DE3) (Novagen). Expression levels and the amount ofsoluble r-44 kDa protein produced were assessed for the differentfragments. This was done following IPTG induction, where by a 1.5 mlcell culture of the recombinant E. coli cell culture was pelleted bycentrifugation and resuspended in 150 ul of binding buffer. Cells werethen sonicated for 10 sec using a microprobe at a setting of 5(Virosonic Digital 475 ultrasonic cell disruptor, The Virtis Company,NY). Following centrifugation for 3 minutes (10,000 rpm) the supernatantwas collected, which represented the soluble fraction. The pellet wasthen washed and the resuspended in binding buffer, which represented theinsoluble fraction. Analysis of the various fractions was carried outusing Western blot analysis and SDS-PAGE. The results of theseexperiments are shown in Table 3. The stability of the r-44 kDa proteinor fragments thereof may also be further enhanced by the site directedmutagenisis of all or selected cysteine residues to serine or alanineresidues.

The 44 kDa adhesin contains six Cys residues that form disulphides whenoxidized which may result in incorrect folding and possibly lead to theformation of insoluble protein. The stability of the r-44 kDa protein orfragments of the r-44 kDa protein may therefore be further enhanced bythe site directed mutagenisis of all or selected cysteine residues toserine or alanine residues.

TABLE 3 Expression levels and solubility of r-44 kD proteins 44 Kd SizeExpression construct Residues (amino acids) levels Solubility Fulllength  1-419 419 +++++ − Fragment 1  2-184 183 +++++ + Fragment 2 2-290 289 +++++ ++ Fragment 3 65-184 120 +++++ +++++ Fragment 4 65-290226 +++++ +++++ Fragment 5 65-418 352 +++++ − Fragment 6 192-290  99+++++ +++++ Fragment 7 192-418  227 +++++ −The amino acid numbering is derived from SEQ ID NO 3.

FIG. 2 shows the results of the full length recombinant 44 kD protein, 2fragments of the 44 kD protein (Fragment 4; residues 65-290 and fragment6; residues 192-290) and a control recombinant protein R2 in the mouseabscess model as described in Example 1. Mice were given 2 doses of 20ug of r-protein 3 weeks apart as in Example 1. Both the full length andthe fragment forms of the 44 kD protein showed statistically significantprotection (p<0.05) compared to the control recombinant protein (R2).Formalin killed whole P. gingivalis (FK-33277) gave complete protectionfrom challenge.

EXAMPLE 3

In addition to using fragments of the 44 kDa adhesin, chimeric proteinsmay be constructed using one or more fragments of the 44 kDa adhesinwith other proteins or protein fragments from other P. gingivalisproteins. Sequence ID 2 and 4 give one such example of a chimericrecombinant protein derived from a fragment of the 44 kDa adhesin(Fragment 6 residues 192-290) linked to another P. gingivalis proteinfragment derived from PG33 (Genbank accession number AF175715) a 95residue C terminal fragment (residues 286-380). In total this chimericprotein has a total of 194 residues.

This chimeric recombinant fusion protein of fragments from the 44 kDaand PG33 proteins was produced by amplifying the PG33 G-terminalfragment by PCR as described in Example I using the following primers.

Forward: (SEQ ID NO.: 31) 5′GGCCCATGGTCGACAATAGTGCAAAGATTGAT 3′ Reverse:(SEQ ID NO.: 32) 5′CTATCCGGCCGCTTCCGCTGCAGTCATTACTACAA 3′

This PCR product was subcloned into the SalI and NotI sites of pET24b togenerate pET24b::PG33C. The 44 kDa fragment 6 PCR product (see example 2for primers) was then subcloned into the EcoRI and Sail of thepET24b::PG33C plasmid to generate a fusion construct of 44 kDa/PG33 i.e.pET24b::PG44f6-PG33C. When this plasmid was transformed into E. colistrain BL21(DE3) and expression studies performed as outlined inExamples 1 and 2, high levels of the chimeric 44 kDa/PG33 recombinantprotein were obtained which was soluble when tested as in Example 2.

EXAMPLE 4

Mouse antisera raised to the recombinant 44 kDa or recombinant fragmentsof the 44 kDa protein react with paraformaldehyde fixed whole P.gingivalis cells indicating that immuno-reactive epitopes are conservedin the recombinant proteins.

Mouse antisera were obtained by immunising BALB/c mice with therecombinant full length 44 kDa protein or with a recombinant fragment ofthe 44 kDa protein as described in Examples 1 and 2. P. gingivalis(strain W50) was anaerobically grown to log phase in brain heartinfusion broth (Oxoid) supplemented with 5 ug/ml hemin and 1 ug/mlvitamin K and 0.5 mg/ml Cysteine. Cells were sedimented bycentrifugation for 15 min at 10,000 rpm at 4° C. and resuspended inphosphate-buffered saline (PBS) containing 1% (wt/vol) paraformaldehyde.Bacteria were placed at 4° C. overnight, then washed and resuspended inPBS to an optical density of 0.25 at OD600 (1×10⁹ cells/ml). Killedbacteria were then mixed in 10 μl aliquots with pooled mouse polyclonalsera at a dilution of 1:100 in 0.22 μm filtered PBS+10% FBS+0.01% Azide(PBS/FA) for 15 min at room temperature. The cells were washed withPBS/FA and were subsequently incubated 15 min with 1 μl of FITC-labelledanti-mouse Immunoglobulin (Silenus) at a dilution of 1:100 in PBS/FA.The cells were then washed and resuspended in 1 ml of PBS/FA.

The fluorescence intensity of stained P. gingivalis cells was quantifiedusing a FACS Calibur-activated fluorescence cell sorter (BectonDickinson) using the 488 nm wavelength band generated from a 15 mW argonion laser. Filtered PBS/FA was used as the sheath fluid. FITC emissionsignals were collected for each analysis which consisted of 20,000 gatedevents that were collected on the basis of size and granularity usingCELLQuest software (Becton Dickinson).

The results are shown in FIG. 3. The % marked on each panel indicatesthe percentage of P. gingivalis cells staining positively i.e., with afluorescence intensity above the background levels seen with no antiseraor with sera from normal mice. All of the recombinant proteins producedantisera that reacted with the majority of P. gingivalis cells althoughantisera to Fragment 4 showed a reduced reactivity compared to the otherr-44 kDa antisera.

EXAMPLE 5

Cloning and expression of the P. gingivalis Kgp39 (Kgp39) and Kgp39fragment (Kgp39frag) adhesin domains in E. coli and testing of therecombinant proteins by ELISA

TABLE 4 Oligonucleotide primers used for the amplificationof the nucleotide sequences encoding Kgp39 Recombinant Protein PrimersKgp39 Forward 5′-GCAGCAGTCGACGCCAACGAAGCCAAGGTTG-3′ (SEQ ID NO.: 33)Reverse 5′-GCAGCACTCGAGGCGCTTGCCATTGGCC-3′ (SEQ ID NO.: 34) Kgp39fragForward 5′-GCAGCAGTCGACTTCTTGTTGGATGCCGATCAC-3′ (SEQ ID NO.: 35) Reverse5′-GCAGCACTCGAGGAATGATTCGGAAAGTGTTG-3′ (SEQ ID NO.: 36)

Kgp39 and Kgp39 fragment adhesin domains were amplified using theprimers listed in Table 4. The primers consist of a 6 nucleotide bufferfollowed by a restriction enzyme site (Sail or XhoI) and sequencespecific for Kgp39. PCR was performed using Taq DNA Polymerase (Promega)under the following conditions: 25 cycles of denaturation (94° C., 45sec), annealing (52° C., 30 sec), and extension (72° C., 60 sec). ThePCR product was ligated into plasmid vector pGEMT-easy (Promega) andtransformed into competent E. coli JM109 (Promega) as previouslydescribed. All procedures were identical for the preparation of bothKgp39 and Kgp39 fragment recombinants and are essentially as describedabove for recombinant Rgp44 fragments. Recombinant plasmidpGEMT-easy-Kgp39 DNA was digested with Sail and XhoI and the purifiedinsert DNA was ligated into purified plasmid expression vector pET28b(Novagen) that had been previously digested with SalI and XhoI. Ligationproducts were transformed into the non-expression host, E. coli JM109and then transformed into the E. coli expression host, HMS174(DE3) aspreviously described. r-Kgp39 expression was induced by addition of IPTGand purified by nickel-affinity chromatography. The integrity of theinsert of pET28b-Kgp39 was confirmed by DNA sequence analysis.

Expression Of Recombinant E. Coli

Recombinant Kgp39 and Kgp39 fragment proteins were expressed byinduction with IPTG using similar methodology as that described forrRgp44 fragments. Briefly, single colony transformants were used toinoculate 5 ml LB containing 50 μg/ml kanamycin at 37° C. on an orbitalshaker overnight. This culture was then used to inoculate 100 ml offresh medium and grown to mid-log growth phase (OD₆₀₀=0.6-1.0) beforeinducing with 0.5 mM IPTG for 6 hours. Cells were then harvested bycentrifugation at 6500×g and stored at −20° C. overnight for theextraction of inclusion bodies.

Isolation and Solubilisation of Inclusion Bodies

The bacterial pellet was thawed on ice and resuspended in 10 mls ofbuffer B (20 mM Na₂HPO₄, 0.5M NaCl, 8M urea). The redissolved cellpellet was sonicated on ice for 3×30 second bursts at 30 secondintervals using a Branson Sonifier® 250 Cell disruptor (BransonUltrasonics Corporation, Danbury, Conn.) with the microtip on setting 3.Insoluble cellular debris was removed by centrifugation at 39000×g for30 minutes at 4° C. and the supernatant collected. The insolublecellular fraction was resuspended in 10 mls of Buffer B. Sodium azide(0.001% v/v) was added to all samples prior to storage at 4° C. Sampleswere then analysed by SDS-PAGE.

Nickel-Nitrilotriaectic Acid (Ni-NTA) Purification and Refolding ofSolubilised Inclusions

Proteins were purified using Pharmacia Biotech HiTrap affinity columns(1 ml) (Amersham Pharmacia Biotech) connected to a Pharmacia FastProtein Liquid Chromatography (FPLC) instrument. The column was coatedwith 5 column volumes of 0.1M NiSO₄ then equilibrated with 10 columnvolumes of Start Buffer (20 mM Na₇HPO₄, 0.5M NaCl, 20 mM imidazole, 8Murea) at a flow rate of 1 ml/min. Samples were loaded onto the column ata flow rate of 0.5 ml/min, then washed with 10 volumes of Start Bufferat a rate of 1 ml/min. Protein was eluted over a linear gradient of 10volumes of Elution Buffer (20 mM Na₂HPO₄, 0.5M NaCl, 200 mM imidazole,8M urea) at a flow rate Of 1 ml/min. Elution fractions were collectedand samples of each fraction were analysed on SDS-PAGE gels aspreviously described.

Renaturation of Recombinant Protein

Removal of 8M urea from the recombinant protein samples was achievedusing Spectrum-Por® Float-A-Lyzer (Alltech, Australia). The molarity ofurea in the samples was taken from 8M initially to 0M over a period of 4days. rKgp39 proteins were refolded by step-wise dialysis from 8 M to 7M to 6 M to 5 M to 4 M to 3 M to 2 M to 1 M to 0.5 M to 0 M Ureacontained in the following buffer: 20 mM Na₂HPO₄, 0.5M NaCl

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISAs were performed to investigate the binding of RgpA-Kgp specificantisera to rKgp39 and rKgp39 fragment and the binding of rKgp39 andrKgp38 fragment to periodontal matrices and host proteins.

Wells of flat-bottomed polyvinyl microtitre plates (Microtitre, DynatechLaboratories, VA, USA) were coated with 5 μg/ml of either rKgp39 orrKgp39 fragment in 0.1M PBS [0.01M Na₂HPO₄, 0.15M NaCl, 1.5 mM KH₂PO₄,3.0 mM KCl, pH 7.4] overnight at room temperature (RT). The coatingsolution was removed and wells were blocked with 1% (w/v) BSA in 0.1MPBST (PBS containing 0.1% (v/v) Tween 20), for 1 hour at RT and plateswashed 4× with 0.1M PBST. Serial dilutions of rabbit antisera directedagainst the P. gingivalis W50 RgpA-Kgp proteinase-adhesin complex(Bhogal et al., 1997) was added to each well and incubated overnight atRT and then washed with 6×PBST. Bound antibody was detected byincubation with horseradish peroxidase-conjugated goat immunoglobulindirected against mouse immunoglobulin (1:4000 dilution) (Sigma, NSW,Australia) in 0.5% (w/v) BSA in 0.1M PBS for 1.5 hr at RT. The plateswere then washed (6×PBST) and substrate [0.9 mM ABTS(2,2′-azino-bis(3-ethylbenz-thiazoline-6-) sulfonic acid], and 0.005%(v/v) H₂O₂, in ABTS buffer (0.1M Na₇HPO₄, 0.08 M citric acidmonohydrate) (100 μl/well) was added. The optical density at 415 nm(OD₄₁₅) was measured by using a Bio-Rad microplate reader (model 450,BioRad, NSW, Australia).

The results are shown in FIG. 4.

Binding of rKgp39 and rKgp39 Fragment to Periodontal Matrices and HostProteins

ELISAs were also performed to investigate the binding characteristics ofrKgp39 and rKgp39 fragment proteins to the host matrix proteinsfibrinogen and collagen type V and to haemoglobin. Microtitre plateswere coated with 10 μg/ml of either fibrinogen, collagen type V orhaemoglobin in 0.1M PBS overnight at RT. The coating solution wasremoved and remaining uncoated plastic was blocked with 2% (w/v) Skimmilk in 0.1M PBST for 1 hr at RT. The blocking solution was removed and5 μg/ml of either rKgp39 or rKgp39 fragment protein in 0.1M PBS wasadded to wells and incubated for 2 hr at RT. Wells were washed 4× with0.1M PBST, then serial dilutions of rabbit anti-RgpA-Kgp complexanti-sera in 1% (w/v) Skim milk in 0.1M PBST was added to each well andincubated overnight at RT. Bound antibody was detected, after washing6×PBST, by incubation with horseradish peroxidase conjugated goatimmunoglobulin directed against rabbit immunoglobulin (1:4000 dilution)(Sigma, NSW, Australia) in 1% (w/v) Skim milk in 0.1M PBST for 1 hr atRT. The plates were developed as described above.

The results are shown in FIGS. 5 and 6.

EXAMPLE 6

This example illustrates that nucleotide sequences encoding RgpA44 orKgp39 or portions thereof, can be inserted into, and expressed byvarious vectors including phage vectors and plasmids. Successfulexpression of the protein and peptides requires that either the insertcomprising the gene or gene fragment, or the vector itself, contain thenecessary elements for transcription and translation which is compatiblewith, and recognized by the particular host system used for expression.DNA encoding the RgpA44 or Kgp39 or fragments thereof (e.g. Example 2),or related peptides or oligopeptides or chimeric peptides can besynthesized or isolated and sequenced using the methods and sequences asillustrated herein. A variety of host systems may be utilized to expressthe RgpA44 or Kgp39 or fragments thereof, related peptides oroligopeptides or chimeras, which include, but are not limited tobacteria transformed with a bacteriophage vector, plasmid vector, orcosmid DNA; yeast containing yeast vectors; fungi containing fungalvectors; insect cell lines infected with virus (e.g. baculovirus); andmammalian cell lines transfected with plasmid or viral expressionvectors, or infected with recombinant virus (e.g. vaccinia virus,adenovirus, adeno-associated virus, retrovirus, etc.).

Using methods known in the art of molecular biology, including methodsdescribed above, various promoters and enhancers can be incorporatedinto the vector or the DNA sequence encoding RgpA44 or Kgp39 amino acidsequences, i.e., related peptides or oligopeptide or chimeras, toincrease the expression of the RgpA44 or Kgp39 amino acid sequences,provided that the increased expression of the amino acid sequences iscompatible with (for example, non-toxic to) the particular host cellsystem used. Thus and importantly, the DNA sequence can consist of thegenes segment encoding the RgpA44 or Kgp39 or fragments thereof, or anyother segment or combined segments of the domain which encode functionaland specific epitopes of the protein. Further, the DNA can be fused toDNA encoding other antigens, such as other bacterial outer membraneproteins, or other bacterial, fungal, parasitic, or viral antigens tocreate a genetically fused (sharing a common peptide backbone)multivalent antigen for use as an improved vaccine composition.

The selection of the promoter will depend on the expression system used.Promoters vary in strength, i.e. ability to facilitate transcription.Generally, for the purpose of expressing a cloned gene, it is desirableto use a strong promoter in order to obtain a high level oftranscription of the gene and expression into gene product. For example,bacterial, phage, or plasmid promoters known in the art from which ahigh level of transcription have been observed in a host cell systemcomprising E. coli include the lac promoter, trp promoter, recApromoter, ribosomal RNA promoter, the P_(R) and P_(L) promoters, lacUV5,ompF, bla, lpp, and the like, may be used to provide transcription ofthe inserted DNA sequence encoding amino acid sequences.

Additionally, if protein, related peptides or oligopeptides or chimerasmay be lethal or detrimental to the host cells, the host cellstrain/line and expression vectors may be chosen such that the action ofthe promoter is inhibited until specifically induced. For example, incertain operons the addition of specific inducers is necessary forefficient transcription of the inserted DNA (e.g., the lac operon isinduced by the addition of lactose or isopropylthio-beta-D-galactoside).A variety of operons such as the trp operon, are under different controlmechanisms. The trp operon is induced when tryptophan is absent in thegrowth media. The P_(L) promoter can be induced by an increase intemperature of host cells containing a temperature sensitive lambdarepressor. In this way, greater than 95% of the promoter-directedtranscription may be inhibited in uninduced cells. Thus, expression ofrecombinant RgpA44 protein, related peptides, or oligopeptides orchimeras may be controlled by culturing transformed or transfected cellsunder conditions such that the promoter controlling the expression fromthe inserted DNA encoding RgpA44 amino acid sequences is not induced,and when the cells reach a suitable density in the growth medium, thepromoter can be induced for expression from the inserted DNA.

Other control elements for efficient gene transcription or messagetranslation include enchancers, and regulatory signals. Enhancersequences are DNA elements that appear to increase transcriptionalefficiency in a manner relatively independent of their position andorientation with respect to a nearby gene. Thus, depending on the hostcell expression vector system used, an enhancer may be placed eitherupstream or downstream from the inserted DNA sequences encoding RgpA44or Kgp39 amino acid sequences to increase transcriptional efficiency. Asillustrated previously in this example, other specific regulatorysequences have been identified which may effect the expression from thegene segment encoding RgpA44 or Kgp39 and related peptides or chimeras.These or other regulatory sites, such as transcription or translationinitiation signals, can be used to regulate the expression of the geneencoding RgpA44 or Kgp39, or gene fragments thereof. Such regulatoryelements may be inserted into DNA sequences encoding RgpA44 or Kgp39amino acid sequences or nearby vector DNA sequences using recombinantDNA methods described herein for insertion of DNA sequences.

Accordingly, P. gingivalis nucleotide sequences containing regionsencoding for RgpA44 or Kgp39, related peptides, or oligopeptides orchimeras can be ligated into an expression vector at a specific site inrelation to the vector's promoter, control, and regulatory elements sothat when the recombinant vector is introduced into the host cell the P.gingivalis-specific DNA sequences can be expressed in the host cell. Forexample, the RgpA44 or Kgp39 specific DNA sequence containing its ownregulatory elements can be ligated into an expression vector in arelation or orientation to the vector promoter and control elementswhich will allow for expression of the RgpA44 or Kgp39 or derivatives.The recombinant vector is then introduced into the appropriate hostcells, and the host cells are selected, and screened for those cellscontaining the recombinant vector. Selection and screening may beaccomplished by methods known in the art including detecting theexpression of a marker gene (e.g., drug resistance marker) present inthe plasmid, immunoscreening for production of RgpA44 or Kgp39 specificepitopes using antisera generated to RgpA44 or Kgp39 specific epitopes,and probing the DNA of the hosts cells for RgpA44 or Kgp39 specificnucleotide sequence using one or more oligonucleotide sequences andmethods described herein.

Genetic engineering techniques may also be used to characterize, modifyand/or adapt the encoded RgpA44 or Kgp39 recombinant or protein. Forexample, site-directed mutagenesis of RgpA44 or Kgp39 or fragmentsthereof to modify one or all Cys residues to Ser or Ala residues may bedesirable to increase the stability and solubility of the recombinantprotein to allow for easier purification and folding. Further, geneticengineering techniques can be used to generate DNA sequences encoding aportion of the amino acid sequence of RgpA44 or Kgp39 in particular,soluble, hydrophilic sequences corresponding to protective epitopes.Restriction enzyme selection may be done so as not to destroy theimmunopotency of the resultant peptide or oligopeptide or chimera.Antigenic sites of a protein may vary in size but can consist of fromabout 7 to about 14 amino acids. Thus, RgpA44 or Kgp39 will contain manydiscrete antigenic sites; therefore, many partial gene sequences couldencode antigenic epitopes of RgpA44 or Kgp39. These sequences can beconstructed and used in an expression system to generate highlyantigenic chimeric peptides or oligopeptides or proteins. Combinationsof two or more peptides may result in increased immunogenicity. Whenusing combinations of antigens these antigens may be related (i.e. fromthe same gene sequence or from a closely related gene from the sameorganism). The antigens may be generated from a related organism (i.e.another oral bacterium present in subgingival plaque), or from a moredistantly-related organism. In particular the host organism for thevector containing the RgpA44 or Kgp39 related genes and constructs canbe a commensal inhabitant of the oral cavity; for example an inhabitantof subgingival plaque, supragingival plaque or a bacterium associatedwith the oral mucosa. Examples of commensal intra-oral bacteria would beStreptococcus species and Actinomyces species, e.g. Streptococcussalivarius, Streptococcus sanguis, Actinomyces naeslundii. Theseorganisms can be isolated from the periodontitis patient and thengenetically engineered to express the RgpA44 or Kgp39 or components,peptides or chimeras. The DNA encoding the RgpA44 or Kgp39, peptides orchimeras could be linked with DNA encoding leader sequences ofextracellular proteins of these commensal intra-oral bacteria. The DNAencoding the RgpA44 or Kgp39 or derivatives could also be linked with,or inserted into, the DNA encoding extracellular proteins to producesecreted fusion proteins. Examples of extracellular proteins that couldbe used to produce fusion proteins with the RgpA44 or Kgp39, components,peptides or chimeras could be the glucosyltranferases (GTF) orfructosyltransferases (FTF). The recombinant organism would be thenre-introduced into the patients oral cavity and once colonised the oralmucosa or teeth would express the RgpA44 or Kgp39, component, peptide,chimera or fusion to stimulate the mucosal associated lymphoid tissue toproduce neutralising antibodies.

The DNA fragment encoding an antigen may be fused to other DNA sequencesto allow for improved expression and/or purification procedures (i.e.DNA sequences cloned into the vector pTrxFus, are expressed as fusionsto the E. coli protein thioredoxin). This linkage imparts thecharacteristics of thioredoxin to the fusion protein which offerssoluble expression of normally insoluble or difficult to expressproteins. After purification, the native protein is released by removalof the entire thioredoxin by digestion with enterokinase. Furthermore,the antigen may be used as a hapten by fusion to other sequences whichmay increase immunogenicity, if the expressed protein or peptide is notimmunogenic.

Another plasmid expression system involves the pUC-derived pTrcHisexpression vector from Invitrogen. This vector allows high-levelexpression of DNA sequences by the presence of the Trc promoter(containing the −35 region of the Trp promoter together with the −10region of the lac promoter) and an rrnB anti-terminator element. ThepTrcHis vectors also contain a copy of the lac1^(q) gene which encodesthe lac repressor protein. Therefore, expression of the recombinantprotein/peptide is induced by addition of 1 mM IPTG (de-repression) toE. coli grown to mid-log phase. The DNA fragment is inserted into themultiple cloning site which is positioned downstream and in frame with asequence that encodes an N-terminal fusion peptide. The N-terminalfusion peptide encodes (from 5′ to 3′); an ATG translation initiationcodon, a series of 6 histidine residues that function as a metal-bindingdomain in the translated protein, a transcript stabilising the sequencefrom gene 10 of phage T7, and an enterokinase cleavage recognitionsequence. Cell culture lysates of cells harbouring the recombinantplasmid are purified by high-affinity binding to Probond™ resin(Invitrogen). Probond™ is a nickel-charged sepharose resin that is usedto purify recombinant proteins containing a poly-histidine bindingdomain. Bound proteins are eluted from the Probond™ resin with eitherlow pH buffer or by competition with imidazole or histidine. Thepolym-histidine leader peptide may be subsequently removed by digestionof the recombinant expressed protein with Enterokinase. Enterokinaserecognizes the endopeptidase recognition sequence that is engineeredbetween the poly-his affinity tag and the multiple cloning site in thevector to allow for cleavage of the poly-His tail away from the proteinof interest. The purified, recombinant protein may then be used in thegeneration of antibodies, vaccines and the formulation of diagnosticassays as discussed.

EXAMPLE 7

Methods for using RgpA44 or Kgp39 specific nucleotide sequences inmolecular diagnostic assays for the detection of P. gingivalis. Thenucleic acid sequences of the present invention can be used in moleculardiagnostic assays for detecting P. gingivalis genetic material. Inparticular, RgpA44 or Kgp39 sequence-specific oligonucleotides can besynthesized for use as primers and/or probes in amplifying, anddetecting amplified, nucleic acids from P. gingivalis. Recent advancesin molecular biology have provided several means for enzymaticallyamplifying nucleic acid sequences. Currently the most commonly usedmethod, PCR™ (polymerase chain reaction Cetus Corporation) involved theuse of Taq Polymerase, known sequences as primers, and heating cycleswhich separate the replicating deoxyribonucleic acid (DNA) strands andexponentially amplify a gene of interest. Other amplification methodscurrently under development include LCR™ (ligase chain reaction,BioTechnica International) which utilizes DNA ligase, and a probeconsisting of two halves of a DNA segment that is complementary to thesequence of the DNA to be amplified; enzyme QB replicase (Gene-TrakSystems) and a ribonucleic acid (RNA) sequence template attached to aprobe complementary to the DNA to be copied which is used to make a DNAtemplate for exponential production of complementary RNA; and NASBA™(nucleic acid sequence-based amplification, Cangene Corporation) whichcan be performed on RNA or DNA as the nucleic acid sequence to beamplified.

Nucleic acid probes that are capable of hybridization with specific genesequences have been used successfully to detect specific pathogens inbiological specimens at levels of sensitivity approaching 10³-10⁴organisms per specimen [1990, Gene Probes for Bacteila, eds. Macario anddeMacario, Academic Press]. Coupled with a method that allows foramplification of specific target DNA sequences, species-specific nucleicacid probes can greatly increase the level of sensitivity in detectingorganisms in a clinical specimen. Use of these probes may allow directdetection without relying on prior culture and/or conventionalbiochemical identification techniques. This embodiment of the presentinvention is directed to primers which amplify species-specificsequences of the gene encoding RgpA44 or Kgp39 of P. gingivalis, and toprobes which specifically hybridize with these amplified DNA fragments.By using the nucleic acid sequences of the present invention andaccording to the methods of the present invention, as few as one P.gingivalis organism may be detected in the presence of 10 ug/mlextraneous DNA.

This embodiment is directed to species-specific oligonucleotides whichcan be used to amplify sequences of P. gingivalis DNA, if present, fromDNA extracted from clinical specimens including subgingival plaque,sputum, blood, abscess and other fluids to subsequently determine ifamplification has occurred. In one embodiment of the present invention,a pair of P. gingivalis-specific DNA oligonucleotide primers are used tohybridize to P. gingivalis genomic DNA that may be present in DNAextracted from a clinical specimen, and to amplify the specific segmentof genomic DNA between the two flanking primers using enzymaticsynthesis and temperature cycling. Each pair of primers are designed tohybridize only to the P. gingivalis nucleotide sequences of the presentinvention to which they have been synthesized to complement; one to eachstrand of the double-stranded DNA. Thus, the reaction is specific evenin the presence of microgram quantities of heterologous DNA. For thepurposes of this description, the primer derived from the sequence ofthe positive (gene) strand of DNA will be referred to as the “positiveprimer”, and the primer derived from the sequence of the negative(complementary) strand will be referred to as the “negative primer”.Amplification of DNA may be accomplished by any one of the methodscommercially available. For example, the polymerase chain reaction maybe used to amplify the DNA. Once the primers have hybridized to oppositestrands of the target DNA, the temperature is raised to permitreplication of the specific segment of DNA across the region between thetwo primers by a thermostable DNA polyinerase. Then the reaction isthermocycled so that at each cycle the amount of DNA representing thesequences between the two primers is doubled, and specific amplificationof the P. gingivalis DNA sequences, if present, results. Furtheridentification of the amplified DNA fragment, as being derived from P.gingivalis DNA, may be accomplished by liquid hybridization. This testutilizes one or more labeled oligonucleotides as probes to specificallyhybridize to the amplified segment of P. gingivalis DNA. Detection ofthe presence of sequence-specific amplified DNA may be accomplishedusing any one of several methods known in the art such as a gelretardation assay with autoradiography. Thus, the nucleotide sequencesof the present invention provide basis for the synthesis ofoligonucleotides which have commercial applications in diagnostic kitsfor the detection of P. gingivalis. In a related embodiment, theoligonucleotides used as primers may be labeled directly, or synthesizedto incorporate label. Depending on the label used, the amplificationproducts can then be detected, after binding onto an affinity matrix,using isotopic or calorimetric detection.

DNA may be extracted from clinical specimens which may contain P.gingivalis using methods known in the art. For example, cells containedin the specimen may be washed in TE buffer and pelleted bycentrifugation. The cells then may be resuspended in 100 ul ofamplification reaction buffer containing detergents and proteinase K.Using the polymerase chain reaction, the resultant sample may becomposed of the cells in 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.01% gelatin, 0.45% NP40™, 0.045% Tween 20™, and 60 ug/ml proteinase K.The sample is incubated in a 55° C. water bath for 1 hour. Following theincubation, the sample is incubated at 95° C. for 10 minutes toheat-inactivate the proteinase K. The sample may then be amplified inaccordance with the protocol for the polymerase chain reaction as setforth below.

The P. gingivalis DNA may be amplified using any one of severalprotocols for amplifying nucleic acids by the polymerase chain reaction.In one mode of this embodiment, the gene encoding the RgpA44 or Kgp39may be amplified from clinical isolates of P. gingivalis using thefollowing conditions. DNA to be amplified (1 mg of genomic DNA) isdistributed to 0.5 ml microfuge tubes and the volume adjusted to 50 ulby adding a reaction mixture comprising 0.2 mM dNTPs (dATP, dCTP dGTP,dTTP), 0.25 ug of each positive and negative oligonucleotide primer, 1unit of TaqI polymerase, TaqI 10× buffer (5 ul), 1 mM MgCl₂ (finalconcentration), and sterile distilled water to achieve the total volume.The TaqI polymerase is added to the reaction mixture just before use andis gently mixed, not vortexed. A layer of mineral oil, approximately 2drops, is added to each tube and then the tubes are placed in thethermal cycler. Thirty to thirty-five cycles are general sufficient forbacterial DNA amplification. One cycle consists of 1 minute at 95° C., 1minute at 37° C., and 1 minute at 72° C. The first cycle includes aminute incubation at 95° C. to assure complete denaturation.

Oligonucleotides useful as primers or probes which specificallyhybridize to the gene encoding the RgpA44 or Kgp39 of P. gingivalis andused in DNA amplification and/or detection can be biochemicallysynthesized, using methods known in the art, from the nucleotidesequences in the Sequence ID listings herein. For detection purposes,the oligonucleotides of the present invention may be end-labeled with aradioisotope. Probe sequences, internal to the two primers used foramplification of the gene sequence, may be end-labeled using T4polynucleotide kinase and gamma ³²P ATP. Twenty pMols of probe DNA inkinase buffer (50 mM Tris, pH 7.6 10 mM MgCl₂, 5 mM dithiothreitol, 0.1mM spermidine-HCl, 0.1 mM EDTA, pH 8.0) is mixed with 120 uCi of gamma³²P ATP and incubated at 37° C. for 1 hour. Labeled probe is separatedfrom unincorporated label on an 8% acrylamide gel run for 1 hour at 200volts in Tris Borate EDTA (TBE) buffer at room temperature. Labeledprobe is first located by exposing the acrylamide gel to x-ray film forthree minutes. The resulting autoradiogram is then positioned under thegel, and the band containing the labeled probe was excised from the gel.The gel slice is pulverized in one milliliter of sterile distilledwater, and the probe is eluted by shaker incubation overnight at 37° C.The eluted probe is separated from the gel fragments by centrifugationusing a chromatography prep column. Radioactivity of the probe isdetermined, by counting one microliter of the labeled probe on a glassfibre filter, by liquid scintillation. Such probe sequences may bechosen from any of the sequences disclosed herein provided the probesequence is internal to the two primers used for amplification of thedesired nucleotide sequence disclosed in the present invention.

Alternative methods known in the art may be used to improve thedetection of amplified target sequences in accordance with thecompositions and methods of the present invention. The sensitivity ofdetection of the amplified DNA sequences can be improved by subjectingthe sequences to liquid hybridization. Alternative methods of detectionknown in the art, in addition to gel electrophoresis and gelelectrophoresis with Southern hybridization and autoradiography, thatmay be used with the compositions and methods of the present inventioninclude: restriction enzyme digestion with gel electrophoresis;slot-blot hybridization with a labeled oligonucleotide probe;amplification with a radiolabeled oligonucleotide probe; amplificationwith a radiolabeled primer with gel electrophoresis, Southernhybridization and autoradiography; amplification with a radiolabeledprimer with dot blot and autoradiography; amplification witholigonucleotides containing affinity tags (ex. biotin, or one primerincorporating biotin and the other primer with a sequence specific for aDNA binding protein) followed by detection in an affinity-based assay(ex. ELISA); and amplification with oligonucleotides containingfluorophores followed by fluorescence detection.

One embodiment of non-isotopic detection involves incorporating biotininto the oligonucleotide primers of the present invention. The 5′-aminogroup of the primers may be biotinylated with sulfo-NHS-biotin, orbiotin may be incorporated directly into the primer by synthesizing theprimer in the presence of biotin-labeled dNTPs. The non-isotopic labeledprimers are then used in amplifying DNA from a clinical specimen. Thedetection for the presence or absence of amplified target sequences maybe accomplished by capturing the amplified target sequences using anaffinity matrix having avidin bound thereto, followed by incubation withan avidin conjugate containing an enzyme which can be used to visualizethe complex with subsequent substrate development. Alternatively, theamplified target sequences may be immobilized by hybridization to thecorresponding probes of the target sequence wherein the probes have beenaffixed onto a matrix. Detection may be accomplished using an avidinconjugate containing an enzyme which can be used to visualize thecomplex with subsequent substrate development.

EXAMPLE 8 Methods for Using RgpA44 or Kgp39, Peptides or ChimericPeptides in Diagnostic Immunoassays.

The RgpA44 or Kgp39 protein, related peptides, oligopeptides or chimerascan be purified for use as immunogens in vaccine formulations; and asantigens for diagnostic assays or for generating P. gingivalis-specificantisera of therapeutic and/or diagnostic value. The RgpA44 or Kgp39from P. gingivalis or oligopeptides or peptides or chimeras thereof, orrecombinant protein, recombinant peptides, or recombinant oligopeptidesproduced from an expression vector system, can be purified with methodsknown in the art including detergent extraction, chromatography (e.g.,ion exchange, affinity, immunoaffinity, or ultrafiltration and sizingcolumns), differential centrifugation, differential solubility, or otherstandard techniques for the purification of proteins.

As used throughout the specification, RgpA44 or Kgp39 oligopeptides aredefined herein as a series of peptides corresponding to a portion of theamino acid sequence of the RgpA44 or Kgp39 respectively as disclosed inthe enclosed sequences that are synthesized as one or chemically-linked.Such peptides or oligopeptides can be synthesized using one of theseveral methods of peptide synthesis known in the art including standardsolid phase peptide synthesis using tertbutyloxycarbonyl amino acids[Mitchell et al., 1978, J. Org. Chem. 43:2845-2852], using9-fluorenylmethyloxycarbonyl amino acids on a polyamide support [Drylandet al., 1986, J. Chem. So. Perkin Trans. I, 125-137]; by pepscansynthesis [Leysen et al., 1987, J Immunol Methods 03:259; 1984; andProc. Natl. Acad. Sci. USA 81:3998]; by standard liquid phase peptidesynthesis; or by recombinant expression vector systems. Modification ofthe peptides or oligopeptides, such as by deletion and substitution ofamino acids (and including extensions and additions to amino acids) andin other ways, may be made so as to not substantially detract from theimmunological properties of the peptide or oligopeptide. In particular,the amino acid sequences of the RgpA44 or Kgp39, or peptide oroligopeptide or chimera thereof, may be altered by replacing one or moreamino acids with functionally equivalent amino acids resulting in analteration which is silent in terms of an observed difference in thephysicochemical behaviour of the protein, peptide, or oligopeptide orchimera. Functionally equivalent amino acids are known in the art asamino acids which are related and/or have similar polarity or charge.Thus, an amino acid sequence which is substantially that of the aminoacid sequences depicted in the Sequence Listing herein, refers to anamino acid sequence that contains substitutions with functionallyequivalent amino acids without changing the primary biological functionof protein, peptide, or oligopeptide or chimera.

Purified RgpA44 or Kgp39 protein, peptides, oligopeptides and chimerasmay be used as antigens in immunoassays for the detection of P.gingivalis-specific antisera present in the body fluid of an individualsuspected of having an infection caused by P. gingivalis. The detectionof RgpA44 or related peptides as an antigen in immunoassays, includesany immunoassay known in the art including, but not limited to,radioinimunoassay, enzyme-linked immunosorbent assay (ELISA), “sandwich”assay, precipitin reaction, agglutination assay, fluorescentimmunoassay, and chen-liluniinescence-based immunoassay.

EXAMPLE 9 Methods and Compounds for Vaccine Formulations Related toRgpA44 or Kgp39 and Related Peptides and Chimeras.

This embodiment of the present invention is to provide recombinantRgpA44 or Kgp39 protein and/or peptides or oligopeptides or chimerasthereof, to be used in as immunogens in a prophylactic and/ortherapeutic vaccine for active immunization to protect against or treatinfections caused by P. gingivalis. For vaccine purposes, an antigen ofP. gingivalis comprising a bacterial protein should be immunogenic, andinduce functional antibodies directed to one or more surface-exposedepitopes on intact bacteria, wherein the epitope(s) are conservedamongst strains of P. gingivalis.

For vaccine development, RgpA44 or Kgp39 specific amino acid sequencesmay be purified from a host containing a recombinant vector whichexpresses RgpA44 or Kgp39 or related peptides or chimeras. Such hostsinclude, but are not limited to, bacterial transformants, yeasttransformants, filamentous fungal transformants, and cultured cells thathave been either infected or transfected with a vector which encodesRgpA44 or Kgp39 amino acid sequences. The recombinant protein, peptide,or oligopeptide or chimera immunogen is included as the relevantimmunogenic material in the vaccine formulation, and in therapeuticallyeffective amounts, to induce an immune response. Many methods are knownfor the introduction of a vaccine formulation into the human or animalto be vaccinated. These include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, ocular,intranasal, and oral administration. The vaccine may further comprise aphysiological carrier such as a solution, a polymer or liposomes; and anadjuvant, or a combination thereof.

Various adjuvants are used in conjunction with vaccine formulations. Theadjuvants aid by modulating the immune response and in attaining a moredurable and higher level of immunity using smaller amounts of vaccineantigen or fewer doses than if the vaccine antigen were administeredalone. Examples of adjuvants include incomplete Freund's adjuvant (IFA),Adjuvant 65 (containing peanut oil, mannide monooleate and aluminummonostrearate), oil emulsions, Ribi adjuvant, the pluronic polyols,polyamines, Avridine, Quil A, saponin, MPL, QS-21, and mineral gels suchas aluminium salts. Other examples include oil in water emulsions suchas SAF-1, SAF-0, MF59, Seppic ISA720, and other particulate adjuvantssuch as ISCOMs™ and ISCOM Matrix™. An extensive but not exhaustive listof other examples of adjuvants are listed in Cox and Coulter 1992 [Wong,W K (ed.) Animals parasite control utilising technonolgy. Bocca Raton;CRC press, 1992; 49-112]. In addition to the adjuvant the vaccine mayinclude conventional pharmaceutically acceptable carriers, excipients,fillers, buffers or diluents as appropriate. One or more doses of thevaccine containing adjuvant may be administered prophylactically toprevent periodontitis or therapeutically to treat already presentperiodontitis.

In another preferred composition the preparation is combined with amucosal adjuvant and administered via the oral route. Examples ofmucosal adjuvants are cholera toxin and heat labile E. coli toxin, thenon-toxic B subunits of these toxins, genetic mutants of these toxinswhich have a reduced toxicity. Other methods which may be utilised todeliver RgpA44 orally include incorporation of the protein intoparticles of biodegradable polymers (such as acrylates or polyesters) bymicroencapsulation to aid uptake of the microspheres from thegastrointestinal tract and to protect degradation of the proteins.Liposomes, ISCOMs™, hydrogels are examples of other potential methodswhich may be further enhanced by the incorporation of targetingmolecules such as LTB, CTB or lectins for delivery of the RgpA44 proteinor peptide to the mucosal immune system. In addition to the vaccine andthe mucosal adjuvant or delivery system the vaccine may includeconventional pharmaceutically acceptable carriers, excipients, fillers,coatings, dispersion media, antibacterial and antifungal agents, buffersor diluents as appropriate.

Another embodiment of this mode of the invention involves the productionof recombinant RgpA44 or Kgp39 specific amino acid sequences as ahapten, i.e. a molecule which cannot by itself elicit an immuneresponse. In such case, the hapten may be covalently bound to a carrieror other immunogenic molecule which will confer immunogenicity to thecoupled hapten when exposed to the immune system. Thus, such a RgpA44 orKgp39 specific hapten linked to a carrier molecule may be the immunogenin a vaccine formulation.

Another mode of this embodiment provides for either a live recombinantviral vaccine, recombinant bacterial vaccine, recombinant attenuatedbacterial vaccine, or an inactivated recombinant viral vaccine which isused to protect against infections caused by P. gingivalis. Vacciniavirus is the best known example, in the art, of an infectious virus thatis engineered to express vaccine antigens derived from other organisms.The recombinant live vaccinia virus, which is attenuated or otherwisetreated so that it does not cause disease by itself, is used to immunizethe host. Subsequent replication of the recombinant virus within thehost provides a continual stimulation of the immune system with thevaccine antigens such as recombinant RgpA44 or Kgp39 protein, relatedpeptides or chimeras, thereby providing long lasting immunity.

Other live vaccine vectors include: adenovirus, cytomegalovirus, andpreferably the poxviruses such as vaccinia [Paoletti and Panicali, U.S.Pat. No. 4,603,112] and attenuated Salmonella strains [Stocker et al.,U.S. Pat. Nos. 5,210,035, 4,837,151 and 4,735,801; and Curtiss et al.,1988, Vaccine 6:155-160]. Live vaccines are particularly advantageousbecause they continually stimulate the immune system which can confersubstantially long-lasting immunity. When the immune response isprotective against subsequent P. gingivalis infection, the live vaccineitself may be used in a preventive vaccine against P. gingivalis. Inparticular, the live vaccine can be based on a bacterium that is acommensal inhabitant of the oral cavity. This bacterium can betransformed with a vector carrying a recombinant RgpA44 or Kgp39,peptides, oligopeptides or chimeric peptides and then used to colonisethe oral cavity, in particular the oral mucosa. Once colonised the oralmucosa, the expression of the recombinant protein, peptide or chimerawill stimulate the mucosal associated lymphoid tissue to produceneutralising antibodies. To further illustrate this mode of theembodiment, using molecular biological techniques such as thoseillustrated in Example 8, the genes encoding the RgpA44 or Kgp39 or genefragments encoding one or more peptides or chimeras may be inserted intothe vaccinia virus genomic DNA at a site which allows for expression ofepitopes but does not negatively affect the growth or replication of thevaccinia virus vector. The resultant recombinant virus can be used asthe immunogen in a vaccine formulation. The same methods can be used toconstruct an inactivated recombinant viral vaccine formulation exceptthat the recombinant virus is inactivated, such as by chemical meansknown in the art, prior to use as an immunogen and without substantiallyaffecting the immunagenicity of the expressed immunogen. A mixture ofinactivated viruses which express different epitopes may be used in theformulation of a multivalent inactivated vaccine. In either case, theinactivated recombinant vaccine or mixture of inactivated viruses may beformulated with a suitable adjuvant in order to enhance theimmunological response to the vaccine antigens.

In another variation of this embodiment, genetic material is useddirectly as the vaccine formulation. Nucleic acid (DNA or RNA)containing sequences encoding the RgpA44 or Kgp39 protein, relatedpeptides or oligopeptides or chimeras, operatively linked to one or moreregulatory elements can be introduced directly to vaccinate theindividual (“direct gene transfer”) against pathogenic strains of P.gingivalis. Direct gene transfer into a vaccinated individual, resultingin expression of the genetic material by the vaccinated individual'scells such as vascular endothelial cells as well as the tissue of themajor organs, has been demonstrated by techniques in the art such as byinjecting intravenously an expression plasmid:cationic liposome complex[Zhu et al., 1993, Science 261:209-211]. Other effective methods fordelivering vector DNA into a target cell are known in the art. In oneexample, purified recombinant plasmid DNA containing viral genes hasbeen used to inoculate (whether parentally, mucosally, or via gene-gunimmunization) vaccines to induce a protective immune response [Fynan etcd. 1993, Proc Natl. Acad Sci USA 90:11478-11182]. In another example,cells removed from an individual can be transfected or electroporated bystandard procedures known in the art, resulting in the introduction ofthe recombinant vector DNA into the target cell. Cells containing therecombinant vector DNA may then be selected for using methods known inthe art such as via a selection marker expressed in the vector, and theselected cells may then be re-introduced into the individual to expressthe RgpA44 or Kgp39 protein, related peptides or oligopeptides orchimeras.

One preferred method of vaccination with genetic material comprises thestep of administering to the individual the nucleic acid molecule thatcomprises a nucleic acid sequence that encodes the RgpA44 or Kgp39protein, related peptides, or oligopeptides or chimeras, wherein thenucleic acid molecule is operatively linked to one or more regulatorysequences necessary for expression. The nucleic acid molecule can beadministered directly, or first introduced into a viral vector andadministered via the vector. The nucleic acid molecule can beadministered in a pharmaceutically acceptable carrier or diluent and maycontain compounds that can enhance the effectiveness of the vaccine.These additional compounds include, but are not limited to, adjuvantsthat enhance the immune response, and compounds that are directed tomodulate the immune response, e.g. cytokines, collectively referred toas “immune modulators”; or other compounds which increase the uptake ofnucleic acid by the cells, referred to as “nucleic acid uptakeenhancers”. The immunization with the nucleic acid molecule can bethrough any parental route (intravenous, intraperitoneal, intradermal,subcutaneous, or intramuscular), or via contact with mucosal surfaces ofthe nasopharynx, trachea, or gastrointestinal tract.

As an alternative to active immunization, immunization may be passive,i.e. immunization comprising administration of purified immunoglobulincontaining antibody against RgpA44 or Kgp39 epitopes.

EXAMPLE 10

The following is a proposed example of a toothpaste formulationcontaining anti-RgpA44 or anti-Kgp39 antibodies.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Glycerol 20.0 Sodiumcarboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroylsarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1 Chlorhexidine gluconate0.01 Dextranase 0.01 Goat serum containing anti-RgpA44 0.2 or anti-Kgp39Water balance

EXAMPLE 11

The following is another proposed example of a toothpaste formulation.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase0.01 Bovine serum containing anti- 0.2 RgpA(788-1004) Water balance

EXAMPLE 12

The following is another proposed example of a toothpaste formulation.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Lauroyl diethanolamide1.0 Sucrose monolaurate 2.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01Bovine milk Ig containing anti- 0.1 RgpA44 Water balance

EXAMPLE 13

The following is another proposed example of a toothpaste formulation.

Ingredient % w/w Sorbitol 22.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0Gantrez 19.0 Water (deionised) 2.69 Sodium Monofluorophosphate 0.76Sodium saccharine 0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavouroil 0.95 anti-RgpA44 mononoclonal 0.3 sodium lauryl sulphate 2.00

EXAMPLE 14

The following is a proposed example of a liquid toothpaste formulation.

Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3Chlorhexidine gluconate 0.01 Ethanol 3.0 Equine Ig containing 0.2anti-RgpA(788-1004) Linolic acid 0.05 Water balance

EXAMPLE 15

The following is a proposed example of a mouthwash formulation.

Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyl,diethanolamide 0.3 Rabbit Ig containing anti-RgpA44 0.2 Water balance

EXAMPLE 16

The following is a proposed example of a mouthwash formulation.

Ingredient % w/w Gantrez S-97 2.5 Glycerine 10.0 Flavour oil 0.4 Sodiummonofluorophosphate 0.05 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.2 Mouse anti-RgpA44 monoclonal 0.3 Water balance

EXAMPLE 17

The following is a proposed example of a lozenge formulation.

Ingredient % w/w Sugar 75-80 Corn syrup 1-20 Flavour oil 1.2 NaF0.01-0.05 Mouse anti-RgpA44 monoclonal 0.3 Mg stearate 1.5 Water balance

EXAMPLE 18

The following is a proposed example of a gingival massage creamformulation.

Ingredient % w/w White petrolatum 8.0 Propylene glycol 4.0 Stearylalcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0Sucrose monostearate 0.5 Chlorohexidine gluconate 0.1 Mouse anti-RgpA44monoclonal 0.3 Water balance

EXAMPLE 19

The following is a proposed example of a chewing gum formulation.

Ingredient % w/w Gum base 30.0 Calcium carbonate 2.0 Crystallinesorbitol 53.0 Glycerine 0.5 Flavour oil 0.1 Rabbit anti-RgpA(788-1004)0.3 monoclonal Water balance

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1.-29. (canceled)
 30. An antigenic composition comprising at least oneisolated and purified protein, wherein the isolated and purified proteinconsists of SEQ ID NO:
 5. 31. The antigenic composition as claimed inclaim 30 wherein the antigenic composition further comprises anadjuvant.
 32. An antigenic composition comprising a chimeric or fusionprotein, wherein the chimeric or fusion protein comprises a first and asecond polypeptide, wherein the first polypeptide consists of SEQ ID NO:5 and the second polypeptide comprises a P. gingivalis sequence.
 33. Theantigenic composition as claims in claim 32, wherein the antigeniccomposition further comprises an adjuvant.
 34. A method of reducing theseverity of P. gingivalis infection in a subject, the method comprisingadministering to the subject an antigenic composition as claimed inclaim
 30. 35. A method of reducing the severity of P. gingivalisinfection in a subject, the method comprising administering to thesubject the antigenic composition as claimed in claim
 32. 36. Anantibody composition, the composition comprising at least one antibody,the antibody being raised against a protein consisting of SEQ ID NO: 5.37. An antibody composition, the composition comprising at least oneantibody which binds a protein consisting of SEQ ID NO: 5, the antibodybeing raised against a chimeric or fusion protein, wherein the chimericor fusion protein comprises a first and a second polypeptide, whereinthe first polypeptide consists of SEQ ID NO: 5 and the secondpolypeptide comprises a P. gingivalis sequence.
 38. An antibodycomposition, the composition comprising at least one antibody, theantibody being raised against a protein, wherein the protein consists ofa sequence selected from the group consisting of SEQ ID NO: 6, SEQ IDNO: 3, residues 1-184 of SEQ ID NO: 3, residues 1-290 of SEQ ID NO: 3,residues 65-184 of SEQ ID NO: 3, residues 65-290 of SEQ ID NO: 3,residues 192-290 of SEQ ID NO: 3, and residues 147-419 of SEQ ID NO: 3.39. A method of reducing the severity of P. gingivalis infection in asubject, the method comprising administering to the subject the antibodycomposition as claimed in claim
 36. 40. A method of reducing theseverity of P. gingivalis infection in a subject, the method comprisingadministering to the subject the antibody composition as claimed inclaim
 37. 41. A method of reducing the severity of P. gingivalisinfection in a subject, the method comprising administering to thesubject the antibody composition as claimed in claim 38.